Sodium ion (Na+) channels, which initiate the action potential in electrically excitable cells, are the molecular targets of local anesthetic drugs. Site-directed mutations in transmembrane segment S6 of domain IV of the Na+ channel alpha subunit from rat brain selectively modified drug binding to resting or to open and inactivated channels when expressed in Xenopus oocytes. Mutation F1764A, near the middle of this segment, decreased the affinity of open and inactivated channels to 1 percent of the wild-type value, resulting in almost complete abolition of both the use-dependence and voltage-dependence of drug block, whereas mutation N1769A increased the affinity of the resting channel 15-fold. Mutation I1760A created an access pathway for drug molecules to reach the receptor site from the extracellular side. The results define the location of the local anesthetic receptor site in the pore of the Na+ channel and identify molecular determinants of the state-dependent binding of local anesthetics.